| The genetic variations of SLA-DQA and DRA genes in Bamei, Juema and Gansu Black pigs were detected via polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP) and DNA sequencing analysis, and further the correlation between the genotypes and haplotypes of DQA and DRA genes and piglet diarrhea were investigated. Physicochemical characteristics, protein structures and functions of DQA and DRA genes coding regions(CDS) in three pig breeds also were predicted through the bioinformatics tools. The main results were shown as below:1. The results showed that exons 2, 3 and 4 of DQA gene from three pig breeds were polymorphic, but there were no polymorphism in exon 1. 8 alleles and 12 genotypes were detected from exon 2, 25 nucleotide including two novel base mutation(c.3979A>G and c.3994T>A) and 15 amino acid substitutions were identified. 3 alleles and 6 genotypes were detected from exon 3, 4 nucleotide substitutions and 3 base deletions were identified, and led to 2 amino acid substitutions and 2 amino acid deletions. 5 alleles and 6 genotypes were detected from exon 4, 9 nucleotide and 4 amino acid substitutions were identified. Expected heterozygosity of exon 4 was greater than observed one in Juema pigs, while expected heterozygosity was less than observed one in all other exons. The chi-square test for Hardy-Weinberg equilibrium disclosed significant differences in genotype distribution of three DQA exons among the three pig breeds(p<0.01).2. The results showed that exons 1, 2 and 4 of DRA gene from three pig breeds were polymorphic, but there were no polymorphism in exon 3. 2 alleles and 3 genotypes were detected from exon 1, only 1 synonymous nucleotide substitution was identified. 2 alleles and 3 genotypes were also detected from exon 2, only 1 nucleotide and acid amino substitution were identified. 5 alleles and 6 genotypes were detected from exon 4, 4 nucleotide and 2 acid amino substitution were identified. Expected heterozygosity of exon 4 in Bamei pigs and exon 2 in Juema and Gansu Black pigs was less than observed one, while Expected heterozygosity was greater than observed one in all other exons. The chi-square test for Hardy-Weinberg equilibrium disclosed significant differences in genotype distribution of DRA exons 1 and 4 among the three pig breeds(p<0.01), while there was no differences in exon 2(p>0.05).3. 37 haplotypes of three DQA exons were identified and frequency of Hp-0.q1 was highest. 19 haplotypes of three DRA exons were identified and frequency of Hp-0.r10 was highest. 13 haplotypes of DQA and DRA exon 2 were identified, frequency of Hp-0.qr1 was maximum, Hp-0.qr11 was minimum. The linkage disequilibrium parameters among exons were low.4. Statistical results revealed a significant correlation between breed and piglet diarrhea(p<0.01), the diarrhea score of Gansu Black was extremely higher than Bamei and Juema pigs, while there was no differences between male and female(p>0.05).5. For DQA exon 2, the genotypic linear contrasts disclosed an extremely high diarrhea score of genotype AA(0.39±0.54), compared to the other genotypes(p<0.01), The scores for genotype CC(-1.31 ± 0.88) were significantly lower than that for genotypes AA and AB(p<0.01) and BB(p<0.05). For DQA exon 3, there was significant differences between genotype and diarrhea(p<0.05), the scores for genotype AC(1.36±0.21) were significantly greater than that for genotypes AA, BB and CC(p<0.01), while genotype AB(1.00±0.16) were significantly greater than genotype CC(p<0.05). For DQA exon 4, genotype was no correlative with diarrhea(p>0.05).6. For DRA exon 1, the genotypic linear contrasts disclosed an extremely high diarrhea score of genotype AB(1.28±0.12), compared to AA and BB(p<0.01). For DRA exon 2, genotypes was no correlative with diarrhea(p>0.05). For DRA exon 4, there was significant differences between genotype and diarrhea(p<0.05), the scores for genotype AA(0.08±0.24) were significantly less than that for genotypes BB, BC and DD(p<0.01) and CD(p<0.05).7. Haplotype of three DQA and DRA exons was significantly associative with diarrhea(p<0.01), the scores of Hp-0.q1(1.00±0.27) and 2(0.97±0.36) were significantly greater than other haplotypes. The score of haplotype Hp-0.r18(1.75±0.17) was significantly higher than Hp-0.r13(p<0.05), and extremely higher than Hp-0.r3、12 and 14(p<0.01). The score of haplotype Hp-0.r14(-0.07±0.37) was significantly less than Hp-0.r01、10、13、15 and 18(p<0.01) and Hp-0.r12(p<0.05). For haplotype of DQA and DRA exon 2, the scores of Hp-0.qr1(1.06 ±0.07) and 2(1.10 ±0.13) were significantly greater than Hp-0.qr3, 4 and 9(p<0.01), while Hp-0.qr9(0.17±0.17) was significantly less than Hp-0.qr1 and 2(p<0.01) and Hp-0.qr3 and 4(p<0.05)8. SLA-DQA gene contained a 767 bp CDS region and encoded a putative 255 amino acids, including 10/11 potential phosphorylation sites and 2 glycosylation sites. The putative secondary structure of DQA was mixed and the percent of random coil was highest. The putative result of DQA protein function showed that the odds of signal transducer, receptor, stress response, immune response and growth factor were higher in Bamei and Gansu Black, while structural protein, transcription and growth factor were higher in Juema. The homologies of DQA amino acid sequences among three pigs and other pig breeds were 96-98 %9. SLA-DRA gene contained a 759 bp CDS region and encoded a putative 252 amino acids, including 11/10 potential phosphorylation sites and 4 glycosylation sites. The putative secondary structure of DRA was mixed and the percent of random coil was highest. The putative result of DRA protein function showed that the odds of signal transducer, receptor, stress response, immune response and growth factor were higher in three pig breeds. The homologies of DRA amino acid sequences among three pigs and other pig breeds were 99 %In conclusion, SLA-DQA was polymorphic, while polymorphism of DRA was lower. Genetic variation in DQA exon 2 and DRA exon 4 were highest. Degree of linkage disequilibrium was low among all exons. The resistance to piglet diarrhea of Bamei and Juema was greater than Gansu Black pigs. DQA exon 2 played a leading role in resistance to piglet diarrhea, and polymorphisms in peptide-binding pockets had a more significant influence on piglet diarrhoea than those in the antigen-binding groove. DRA exon 4 alse had a most significant influence on piglet diarrhoea in DRA coding region. Haplotype of three DQA and DRA genes was significantly associative with diarrhea, Linkage disequilibrium among exons in the same gene had no influence to diarrhea, while Linkage disequilibrium between exon 2 of DQA and DRA had influence to diarrhea. |
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